Immature T lymphocytes differentiating in the thymus make gene products which are found in no other normal tissue. These include a DNA polymerase, terminal deoxynucleotidyl transferase (TdT) and, in mice, the glycoprotein TL. Besides appearing transiently in normal lymphopoiesis, TdT and TL are produced by many leukemias, sometimes giving phenotypes with no normal analogue. We wish to study the molecular mechanisms that regulate expression of these products in normal development and the ways these are altered in malignant cells. Therefore, we propose to develop recombinant plasmids that will serve as hybridization probes for the RNA species that encode TL and TdT. Genomic DNA clones that include a TL structural gene have been made available to us. To use any of these as a probe to measure TL mRNA expression, it is necessary to isolate TL-specific sequences from the majority of the coding sequence which cross-hybridizes extensively with other class I genes. We are subcloning fragments derived from the TL gene and screening them for reaction with RNA species unique to TL+ thymus tissue. TL-specific polyadenylated RNAs have been detected with these probes in both TL+ tumor cell lines and normal thymus tissue. In each case, three distinct RNAs are found, with approximate lengths of 3.1, 1.6, and 1.1 kb. All three hybridize with probes from both the 5' and 3' portionsof the gene. None are expressed in genetically TL- thymus tissue or in phenotypically TL- tissue, such as spleen, from TL+ mice. We are now investigating which species encodes authenticTL protein. To obtain a probe for TdT RNA, we are attempting to use a prokaryotic expression vector (lambda gt11) and an immunological screening technique to detect TdT sequences expressed by the recombinants. Probes so identified will be used to screen a genomic mouse DNA library in lambda Charon 4A (provided by L. Hood). We have developed conditions for efficient gene transfer into T cells so that the full TdT gene can be identified by expression of the TdT protein in recipient cells. The cloned probes will be used on Northern blots to identify TL and TdT RNAs from tumor cells of different phenotypes and from separated subpopulations of normal thymocytes. (M)